Project Summary: Glutamine is a conditionally essential amino acid that has myriad uses in the cell. Aside from direct incorporation into protein, glutamine can be metabolized to generate nucleotides, other amino acids, ATP after anaplerosis into the TCA cycle, and glutathione to protect against oxidative stress. It is unknown how osteoblasts utilize glutamine and when it is required during differentiation. We recently identified glutaminase (GLS) as a critical regulator of Wnt dependent pathological bone formation. GLS deaminates glutamine to form glutamate, the first step in glutamine metabolism. Using a pharmacological approach, we demonstrated GLS activity was required for bone formation in the LRP5 human high bone mass disease mouse model. Importantly, GLS inhibition did not affect bone mass in otherwise wild type mice indicating GLS and glutamine metabolism may not be required for physiological bone formation in mice. In this proposal, we will 1) establish the necessity and sufficiency of glutaminase (GLS) to regulate osteoblast differentiation and bone formation, 2) determine if the molecular regulation of GLS activity by WNT, and 3) understand how glutamine is metabolized in mesenchymal stem cells. Our findings will have broad implications in bone development, maintenance of bone mass, skeletal repair and regeneration, as well as identify if stimulating glutamine metabolism is a viable osteoanabolic target to stimulate bone formation.